Team:Heidelberg/Notebook measure/NotebookRT

Real-time RT-PCR

Contents

8-24-2009

• Design primers and probes for housekeeping genes (GAPDH, G6PD, 18s rRNA, TUBG2, ATCB) and fluorescent proteins (eGFP, mCherry).

8-26-2009

• RNA extraction test (manually).
  • material and methods:
    • Sample: HeLa cells: 10⁶ cells/tube
    • RNeasy Plant Mini Kit
    • Protocol: Purification of Total RNA from Animal Cells Using Spin Technology
  • Predicted yield (see "RNeasy Mini Handbook"-Qiagen): 15 µg/10⁶ cells
  • Experimental yield (acquired with Nanodrop):
    • sample 1: 302 ng/µl, 18.12 µg/10⁶ cells
    • sample 2: 181 ng/µl, 10.86 µg/10⁶ cells

8-31-2009

• RNA extraction (QIAcube)
  • material and methods:
    • Sample: HeLa cells (20 hours after transfection): 0.8*10⁴ cells/tube
    • QIAcube
    • RNeasy Plant Mini Kit
    • Protocol: RNeasy Mini Animal Cell QIAshredder Version 2
  • Yield: not detectable for Nanodrop

9-02-2009

• Primers and probes diluted to 100 µM
• RNA extraction (QIAcube)
  • material and methods:
    • Sample: HeLa cells (with and without DMSO): 10⁶ cells/tube
    • QIAcube
    • RNeasy Plant Mini Kit
    • Protocol: RNeasy Mini Animal Cell QIAshredder Version 2
  • Yield:
    • Sample 1 (without DMSO): 327.7 ng/µl, 16.39 µg/10⁶ cells
    • Sample 2 (with DMSO): 56.2 ng/µl, 2.31 µg/10⁶ cells

9-03-2009

• Primers diluted to 5 µM
• test PCR of primers

9-04-2009

• RNA extration of HeLa transfected with CMV and JeT.
  • yield: 3.7 µg/1.5*10⁵ cells

Gel electrophoresis of RNA extracted from HeLa cells. 1% agarose gel, 100V, 60 min.

• Real-time RT PCR
  • result:mRNA(CMV):mRNA(JeT)=1.73:1

9-10-2009

• RNA extraction of Standard from HeLa cells using QIAcube
  • yield: 29 µg/2.4*10⁶ cells
  • dilution: 20 ng/µl

9-21-2009

• RNA extraction of HeLa cells using QIAcube
  • 12 samples, 1.5*10⁵ cells/tube
  • Yield: <10 ng/µl
  • gel electrophoresis: negative

9-24-2009

• RNA extraction of HeLa cells transfected with CMV and JeT using QIAcube
  • 12 samples each, 6.6*10⁵ cells/tube
  • yield: CMV: 247.9 ng/µl, JeT: 238.3 ng/µl
• Dilution: to 20 ng/µl
• gel electrophoresis: positive

Gel electrophoresis of RNA extracted from HeLa cells. 1% agarose gel, 100V, 60 min.

9-25-2009

• real-time Reverse Transcription PCR for CMV and JeT (20h)

9-30-2009

• RNA extraction of HeLa cells 50 hours after transfected with CMV and JeT

10-02-2009

• NanoDrop the RNA extraction products
  • Dilute the products to 20 ng/µl
• Real-time RT PCR for CMV and JeT (50h)